Heterotrimeric G protein-mediated signal transduction is a tightly regulated event. All known G protein-coupled receptor (GPCR) mediated signaling pathways rely on multiple regulatory mechanisms in order to prevent inappropriate induction of the signal and to facilitate recovery during chronic stimulation (Gilman (1987) Ann. Rev. Biochem. 56:615-649, reviewed in Simon et al. (1991) Science 252:802-808; Conklin and Bourne (1993) Cell 73:631-64 1; Neer (1995) Cell 80:249-257; Rens-Domiano and Hamm (1995) FASEB J. 9:1059-1066). These regulatory mechanisms function at every level of the signaling cascade. Regulation of GPCR activation is believed to involve phosphorylation of the receptor C-terminus and subsequent receptor internalization (Palczewski and Benkovic (1991) Trends Biol. Sci. 16:387-391; Goodman et al. (1996) Nature 383:447-450; Chen and Konopka (1996) Mol. Cell. Biol. 16:247-257), though this does not appear to be a universal mechanism (Daunt et al. (1997) Mol. Pharm. 51:711-720). Known mechanisms of regulation of signal transduction at the level of the heterotrimeric G protein include receptor-mediated facilitation of GTP/GDP exchange on Gxcex1 (reviewed in Simon et al. (1991) Science 252:902-808; Conklin and Bourne (1993) Cell 73:631-641; Neer (1995) Cell 80:249-257; Rens-Domiano and Hamm (1995) FASEB J. 9:1059-1066) and enhancement of the intrinsic GTPase activity of Gxcex1 proteins by RGS-like proteins (reviewed in Berman and Gilman (1998) J. Biol. Chem. 273:1269-1272). Activation of PAKs, serine/threonine kinases that transduce signals from heterotrimeric G proteins to the MAP kinase cascade, has been shown to occur through interaction with either the small G proteins Cdc42 and Rac, or through interaction with heterotrimeric G proteins (reviewed in Sells and Chernoff (997) Trends Cell Biol. 7:162-167). GPCR-coupled MAP kinase cascades and their downstream transcription factors, in nun, are regulated through phosphorylation/dephosphorylation cycles that may or may not require small G proteins (reviewed in Cobb and Goldsmith (1995) J. Biol. Chem. 270:14843-14846). Non-receptor activators of G-proteins have also been identified. These include both protein activators (Strittmatter et al. (1993) Proc. Nat""l Acad. Sci., USA 90:5327-5331; Okamoto et al. (1995) J. Biol. Chem. 270:4205-4208; Sato et al. (1996) J. Biol. Chem. 271:30052-30060) and non-protein activators (summarized in Odagaki et al. (1998) Life Sciences 62:1537-1541.
Even with the identification of these diverse regulatory systems, an in-depth understanding of the temporal and spatial regulation of GPCR mediated signaling remains elusive. In fact, cellular variations in the efficiency and/or specificity of coupling observed for many specific receptor-heterotrimeric G protein complexes suggest the presence of additional unidentified, cell-specific regulators of the signaling process.
In an attempt to identify novel factors involved in regulating signaling through GPCR-mediated signal transduction pathways, a screening system was devised in the yeast Saccharomyces cerevisiae designed to identify receptor-independent activators of the pheromone response pathway. These functional screens can be designed to target not only specific regulatory pathways in yeast, but also an introduced mammalian component or components. Yeast strains containing an intact signal transduction cascade but lacking a functional GPCR were made conditional for growth upon either pheromone pathway activation (activator screen) or pheromone pathway inactivation (inhibitor screen). In addition, the activator yeast strain carries an integrated FUS1p-HIS3 construct, making histidine prototrophy conditional upon pheromone pathway activation. The inhibitor yeast strain carries an episomal FUS1p-CAN1 construct Adult human liver cDNAs expressed in a yeast vector under control of a galactose-inducible promoter were introduced into these strain, and those cDNAs that conferred growth in a galactose- and insert-dependent fashion were identified. Provided herein is the characterization of one fmember of these activator cDNAs, encoding a protein of 281 amino acids with significant homology to ras-related G proteins and containing alterations in conserved amino acids consistent with a deficiency in GTP hydrolysis activity (i.e., a constitutively active form of ras-related G protein). Genetic epistasis tests in yeast were consistent with this activator functioning at the level of the heterotrimeric G-protein and facilitating GTP exchange on the chimeric Gxcex1i2. This protein is referred to herein as Activator of G protein Signaling, or xe2x80x9cAGSxe2x80x9d, or AGS1AGS1 also shows Gxcex1 selectivity, as measured by growth assays in yeast expressing various mammalian Gxcex1 constructs, and tissue specific expression, as measured by Northern blot analysis. A cDNA product identified from the inhibitor screen encodes a previously identified regulator of G-protein signaling, human RGS5.
In one embodiment, an isolated nucleic acid molecule of the present invention encodes an AGS protein (e.g., the nucleic acid molecule has a nucleotide sequence having at least 86% identity to the nucleotide sequence of SEQ ID NO: 1 or the complement thereof). In another embodiment, an isolated nucleic acid molecule of the present invention has a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3, or the complement of SEQ ID NO: 1 or SEQ ID NO: 3. In yet another embodiment, the isolated nucleic acid molecule has the nucleotide sequence of SEQ ID NO: 1, or the complement thereof. In another embodiment, the isolated nucleic acid molecule has the nucleolide sequence of SEQ ID NO: 3, or the complement thereof. In yet another embodiment, an isolated nucleic acid molecule of the present invention encodes a protein that activates G protein-coupled signal transduction in a G protein-coupled receptor independent manner.
In another embodiment, an isolated nucleic acid molecule of the present invention has a nucleotide sequence which encodes a protein having an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO: 2. In another embodiment, the isolated nucleic acid molecule encodes a protein having the amino acid sequence of SEQ ID NO: 2. In yet another embodiment, an isolated nucleic acid molecule of the present invention encodes a protein which activates G protein-coupled signal transduction in a G protein-coupled receptor independent manner.
The present invention also provides vectors including nucleic acid sequences which encode all or a portion of an AGS protein as well as host cells including such vectors. The invention further provides methods for producing an AGS protein including culturing host cells which express an AGS protein. The invention also provides transgenic animals which contain cells carrying a transgene encoding AGS protein.
In another embodiment, the present invention provides isolated AGS proteins (e.g., an isolated AGS protein having an amino acid sequence at least 97% identity to the amino acid sequence of SEQ ID NO: 2.) In another embodiment, the protein has the amino acid sequence of SEQ ID NO: 2. The present invention also provides fusion proteins having at least a portion of an AGS protein operatively linked to a non-AGS polypeptide as well as antibodies that specifically bind to an AGS protein (e.g., monoclonal or polyclonal antibodies). The invention further provides pharmaceutical compositions including AGS proteins or AGS-antibodies.
The present invention further provides methods for identifying compounds that modulate cellular signal transduction. In one embodiment, the method includes the steps of (a) contacting a cell that expresses an AGS protein with a test compound; (b) determining the effect of the test compound on the activity of the AGS protein; and (c) identifying the test compound as a modulator of signal transduction based on the ability of the compound to modulate the activity of the AGS protein in the cell. In another embodiment, the AGS proteins utilized in the subject methods have an amino acid sequence which is at least 97% identical to SEQ ID NO: 2 and stimulates G protein activity in a receptor-independent manner. In yet another embodiment, the AGS protein used in the subject methods has the amino acid sequence of SEQ ID NO: 2.
In yet another embodiment, the compound identified by the above method is a nucleic acid encoding a polypeptide capable of inhibiting the activity of the AGS protein. In still another embodiment, the above method further comprises a nucleic acid encoding an inhibitor of the AGS protein. In a preferred embodiment, the above method is suitable for identifying a test compound capable of modulating the activity of the AGS protein by modulating the inhibitor of the AGS protein.
In a preferred embodiment, cells used in the screening methods of the present invention have been engineered to express the AGS protein. Preferred cells for use in the screening methods are yeast cells. In another preferred embodiment, the yeast cells have further been engineered to express a G protein xcex1 subunit, a chimeric G protein a subunit, or a Gpa1-Gxcex1i2 chimeric G protein xcex1 subunit. The activity of a test compound on a cell-associated activity (e.g., a G-protein mediated activity) can be determined by monitoring a pheromone response pathway in the yeast cells (e.g., by measuring the activity of a pheromone responsive promoter in the yeast cells), by monitoring the ability of the test compound to bind to the AGS protein, or by monitoring the ability of the test compound to modulate the interaction of the AGS protein with a target molecule (e.g., a G protein).
The present invention further provides methods for modulating G protein-coupled signal transduction in a cell (e.g., by contacting a cell with an agent which modulates AGS protein activity or AGS nucleic acid expression). Methods for treating a subject having a disorder characterized by aberrant AGS protein activity or nucleic acid expression are also provided as well as methods for detecting the presence of AGS in a biological sample.
Other features and advantages of the invention will be apparent from the following detailed description and claims.